The long-term objective of this project is to elucidate the regulatory network which controls the coordinated expression of the large number of genes in the nucleus and mitochondrial DNA required for mitochondrial replication. The role of three molecular regulatory signals will be investigated: heme, 3',5'-cyclicAMP and copper ions. The means by which these molecules are detected by the genes will be studied employing genetic modification by traditional methods and with recombinant DNA. It is anticipated that expanded knowledge of normal cellular regulatory mechanisms will be generally useful in understanding those diseases in which cell regulation becomes abnormal. One specific aim is to characterize mutations which alter intracellular transport of 5-aminolevulinate (ALA) synthase, the first enzyme of heme biosynthesis, to determine whether they alter receptors on the mitochondrial membrane. Cloned ALA-synthase gene will be used to determine structure of addressing signals in the pre-enzyme which recognize mitochondrial receptors. A second aim is to determine the regulatory role of the rate-limiting enzyme of yeast heme biosynthesis, ALA-dehydratase. The cloned gene for this enzyme and that for copper chelatin will be utilized together with mutations which render yeast porhyric. Regulatory changes will be monitored by their effects on porphyrin accumulation. The influence of cAMP will be assessed similarly using mutations which alter adenylate cyclase and cAMP-dependent protein kinase. A final objective is to understand the mechanism by which a fraction of the porphyrin produced is shunted into the heme a branch of the tetrapyrrole pathway. Mutants affecting this process will be characterized and the respective normal genes will be cloned.